Rabies is a disease that can occur in all warm-blooded species and is caused by rabies virus. Infection with rabies virus followed by the outbreak of the clinical features in nearly all instances results in death of the infected species. Rabies virus is a non-segmented negative-stranded RNA virus of the Rhabdoviridae family. Rabies virus virions are composed of two major structural components: a nucleocapsid or ribonucleoprotein (RNP), and an envelope in the form of a bilayer membrane surrounding the RNP core. The infectious component of all Rhabdoviruses is the RNP core which consists of the RNA genome encapsidated by the nucleocapsid (N) protein in combination with two minor proteins, i.e. RNA-dependent RNA-polymerase (L) and phosphoprotein (P). The membrane surrounding the RNP core consists of two proteins: a trans-membrane glycoprotein (G) and a matrix (M) protein located at the inner site of the membrane.
The G protein, also referred to as spike protein, is responsible for cell attachment and membrane fusion in rabies virus and additionally is the main target for the host immune system. The amino acid region at position 330 to 340 (referred to as antigenic site III) of the G protein has been identified to be responsible for the virulence of the virus, in particular the Arg residue at position 333. All rabies virus strains have this virulence determining antigenic site III in common.
Conventional Rabies Vaccines for companion animals comprise inactivated rabies plus adjuvants, which are well-known in the art, are diverse in nature. Adjuvants may, for example, consist of water-insoluble inorganic salts, liposomes, micelles or emulsions, i.e. Freund's adjuvant. Other adjuvants may be found in Vogel and Powell, 1995, mentioned supra. Although there is no single mechanism of adjuvant action, an essential characteristic is their ability to significantly increase the immune response to a vaccine antigen as compared to the response induced by the vaccine antigen alone (Nossal, 1999, supra; Vogel and Powell, 1995, supra). In this regard, some adjuvants are more effective at augmenting humoral immune responses; other adjuvants are more effective at increasing cell-mediated immune responses (Vogel and Powell, 1995, supra); and yet another group of adjuvants increase both humoral and cell-mediated immune responses against vaccine antigens (Vogel and Powell, 1995, supra). In sum, adjuvants generally appear to exert their effects in at least one of five ways: 1) facilitate antigen uptake, transport and presentation in the lymph nodes, 2) prolong antigen presentation, 3) signal pathogen-recognition receptors (PRRs) expressed on innate immune cells, 4) cause damage or stress to cells, which signals an immune response, and 5) induce a preferential Th1 or Th2 response (Schijns V E et al. 2007). The immunogenicity of antigens may also be enhanced by the use of genetic adjuvants, such as ligands for receptor residing on immune cell membranes. Genetic adjuvants for DNA vaccines have been reviewed (see, e.g., Calarota & Weiner, Expert Rev Vaccines. 2004 August; 3(4 Suppl): S 135-49, Calarota & Weiner, Immunol Rev. 2004 June; 199:84-99 and Kutzler & Weiner, J Clin Invest. 2004 November; 1 14(9):1241-4), however genetic adjuvants for viral vaccines, especially for poxvirus-based viral vaccines, remain less well-studied.
Several members of tumor necrosis factor superfamily (TNFSF) and their corresponding receptors (TNFRSF) have been shown to provide critical costimulatory signals for immune response (Watts T H. Annu Rev Immunol 2005; 23:23-68). OX40 Ligand (OX40L), also known as gp34, CD252, CD134L or TNFSF4, is a member of the TNF superfamily. Human OX40L shares 46% amino acid sequence identity with its mouse counterpart. Similar to other TNF superfamily members, membrane-bound OX40 Ligand exists as a homotrimer. OX40L binds to OX40 (CD134), a member of the TNF receptor superfamily. OX40 is expressed on activated T cells, while its ligand, OX40L is induced on activated antigen-presenting cells (APCs), such as B cells, and dendritic cells (DCs) [Watts T H. 2005 supra, Sugamura K, et al., Nat Rev Immunol 2004; 4(6):420-31]. OX40-OX40L interaction can promote proliferation, differentiation, and especially survival of CD4+ T cells (Rogers P R, et al., Immunity 2001; 15(3):445-55; Song J, et al., Nat Immunol 2004; 5(2):150-8). Ligation of OX40 has been shown to enhance ex vivo human CD8+ T cell recall responses against viruses, including HIV-1, Epstein-Barr virus (EBV), and influenza virus (Serghides L, et al., J Immunol. 2005; 175(10):6368-77;). Co-immunization of mice with OX40L-expressing canarypox and HIV-1 canarypox vaccine, vCP1452, augmented HIV-1 specific CD8+ T cell responses in terms of frequency and cytokine expression (Liu J. et al., Vaccine. 2009; 275077-5084). However, OX40L did not enhance antibody responses elicited by the HIV-1 canarypox vaccine, suggesting that, canarypox vectors expressing OX40L can enhance the cellular but not humoral immunogenicity of HIV-1 canarypox vaccines. Liu J. et al., 2009, supra).
In the instant disclosure, the OX40L is co-expressed together with rabies G by the same recombinant as opposed to previous works by Serghides L, et al., 2005, supra, where adenovirus-expressed OX40L was used in combination with influenza peptides in an in vitro studies or the work described by Liu J. et al., 2009, where OX40L-expressing canarypox and HIV-1 expressing canarypox were co-administered. Surprisingly, this co-expression of OX40L resulted in 2- to 3-fold increase in peak anti-rabies neutralizing antibody titers as opposed to absence of improvement in humoral immunogenicity in the work reported by Liu J. et al., 2009, supra.
A genetically-adjuvanted Rabies vaccine for companion animals would be highly desirable, as it could avoid or reduce the negative consequences currently associated with conventional chemically adjuvanted vaccines (e.g. injection site reactions, discomfort, pain, non-specific immune responses, increased cancer risk etc.). For example, in cats, vaccine-associated sarcomas have been reported to develop in association with administration of some adjuvanted vaccines. Thus, there is a need for an effective and safe viral vaccine, especially with respect to expression of a target antigen, epitope, immunogen, peptide or polypeptide of interest in an amount sufficient to elicit a protective response.